Studies designed to determine the sequence of the human genome, as well as studies designed to compare human genomic sequences, have elicited information regarding polymorphisms of such sequences. A wide variety of polymorphisms in the human genome have previously been described. The various types of human genetic polymorphisms include single base substitutions, insertions, or deletions; variable numbers of tandem repeats; deletions of all or a large part of a gene; gene amplifications; and chromosomal rearrangements.
Cytochrome P450 (CYP) is a family, or group, of genes in the human genome that encode enzymes several of which facilitate the metabolism of various drugs. One of these genes, CYP2D6, plays a role in the metabolism of a large number of drugs, including several products used to treat psychiatric and cardiovascular disorders. Not surprisingly therefore, some variants of CYP2D6 have been found, at least in part, to alter an individual's ability to metabolize drugs.
While some CYP2D6 polymorphisms have little effect on an individual's ability to metabolize drugs, others have a significant effect. For example, a variant known as CYP2D6 star five (CYP2D6*5, hereinafter *5) comprises a deletion of most of the CYP2D6 gene. *5 is one of several CYP2D6 variants that can contribute to a poor metabolizer phenotype, characteristic of persons having an at least impaired ability to metabolize certain classes of drugs. A possible consequence of the poor metabolizer phenotype is that drugs, normally metabolized by CYP2D6, may build up to toxic concentrations in poor metabolizer individuals. Alternatively, a drug requiring activation by CYP2D6 protein may not be efficacious in persons having the poor metabolizer phenotype. Other variants that can contribute to a poor metabolizer phenotype include a single nucleotide substitution (CYP2D6 star 4 or CYP2D6*4, herenafter *4) and two single nucleotide deletions (CYP2D6 star 3 or CYP2D6*3 hereinafter *3; and CYP2D6 star 6 or CYP2D6*6 hereinafter *6).
On the other hand, some individuals carry multiple copies of the CYP2D6 gene (variously referred to as “an amplification” of the CYP2D6 gene or CYP2D6×2, hereinafter ×2) in their genomes. Individuals with this variant may have an increased ability to metabolize certain classes of drugs and therefore normal doses of these drugs are cleared from the body quite quickly and have little chance to achieve the desired effect. Other variants of CYP2D6 including CYP2D6 star 2 (CYP2D6*2 hereinafter *2), a single nucleotide substitution, and CYP2D6 star 9 (CYP2D6*9 hereinafter *9), a three nucleotide deletion, have not been demonstrated to have any affect on an individual's ability to metabolize drugs. Hence, there are various and different types of CYP2D6 variants that may or may not impair drug metabolism in humans.
Many different methods have been proposed to detect variants such as those mentioned above. Unfortunately, however, different detection methodologies have previously appeared necessary to detect different types of variants. While nucleic acid amplification based assays for single nucleotide polymorphisms have used technology that is amenable to automation, amplification based assays for detecting larger variations such as large deletions or insertions are not readily amenable to automation. For example, “allele specific PCR” is described in European Patent Application 463 395 and is a method for detecting single nucleotide polymorphisms. Allele specific PCR based assays can be performed using methodologies that are relatively easy to automate. On the other hand, “long PCR” has been employed to detect large insertions or deletions of nucleic acid sequences, particularly *5 and ×2 (Johansson I., Lundqvist E., Dahl M. L., and Ingelman-Sundberg, Pharmacogenomics, 6, 351–355 (1996). While amplification products from allele specific PCR and long PCR can be detected on gels, long PCR products are somewhat limited to gel detection. Accordingly, current methodologies require the use of gels to detect certain types of mutations.
It is well known, however, that running gels is time consuming and therefore expensive. Moreover, there is no single platform that enables the detection of, for example, single base polymorphisms and large deletions using a single format that is readily amenable to automation. Accordingly, there is a need for means to detect amplification products from multiple and different types of polymorphisms on a single automated platform.